The central goal of this Program is to define the functions of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) and the way in which its functions are subverted by genetic mutations that cause cystic fibrosis. This will be accomplished by examining the interactions between CFTR, C1 transport and the trafficking of cell membranes and membrane proteins. We will express CFTR in epithelial cells and nonepithelial cells and use a variety of functional assays for C1 channel activity and membrane cycling to resolve CFTR's functions. At the biochemical level, nucleotide binding, ATP hydrolysis and kinase- mediated phosphorylation of CFTR and CFTR domains will be determined after expressing and purifying CFTR. We will determine the C1 and solute transport properties of internal membrane vesicles containing CFTR. At the level of cell biology, we will determine the location of CFTR and changes in its location during stimulation of C1 conductance by CAMP and Ca. We will determine whether membrane fusion is necessary for activation of plasma membrane C1 conductance. The role of the cytoskeleton in C1 channel activation and membrane trafficking will be determined. At the level of cell physiology, we will identify the single-channel basis of the CAMP-activated C1 conductance in secretory epithelial cells. We will compare the properties and regulation of C1 channels in absorptive and secretory epithelia. Three studies will identify the functions of CFTR and elucidate novel mechanisms of epithelial cell regulation. A complete understanding of the cell physiology of CFTR can elucidate new and optimal therapies that will improve the lives of CF patients.